Pseudopolymorphic Forms Of A HIV Protease Inhibitor

ABSTRACT

New pseudopolymorphic forms of (3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl(1S,2R)-3-[[(4-aminophenyl)sulfonyl](isobutyl)amino]-1-benzyl-2-hydroxypropylcarbamate and processes for producing them are disclosed.

TECHNICAL FIELD

This invention relates to novel pseudopolymorphic forms of(3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl(1S,2R)-3-[[(4-aminophenyl)sulfonyl](isobutyl)amino]-1-benzyl-2-hydroxypropylcarbamate,a method for their preparation as well as their use as a medicament.

BACKGROUND OF THE INVENTION

Virus-encoded proteases, which are essential for viral replication, arerequired for the processing of viral protein precursors. Interferencewith the processing of protein precursors inhibits the formation ofinfectious virions. Accordingly, inhibitors of viral proteases may beused to prevent or treat chronic and acute viral infections.(3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl(1S,2R)-3-[[(4-aminophenyl)sulfonyl](isobutyl)amino]-1-benzyl-2-hydroxypropylcarbamatehas HIV protease inhibitory activity and is particularly well suited forinhibiting HIV-1 and HIV-2 viruses.

The structure of(3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl(1S,2R)-3-[[(4-aminophenyl)sulfonyl](isobutyl)amino]-1-benzyl-2-hydroxypropylcarbamate,is shown below:

Compound of formula (X) and processes for its preparation are disclosedin EP 715618, WO 99/67417, U.S. Pat. No. 6,248,775, and in Bioorganicand Chemistry Letters, Vol. 8, pp. 687-690, 1998, “Potent HIV proteaseinhibitors incorporating high-affinity P₂-igands and(R)-(hydroxyethylamino)sulfonamide isostere”, all of which areincorporated herein by reference.

Drugs utilized in the preparation of pharmaceutical formulations forcommercial use must meet certain standards, including GMP (GoodManufacturing Practices) and ICH (International Conference onHarmonization) guidelines. Such standards include technical requirementsthat encompass a heterogeneous and wide range of physical, chemical andpharmaceutical parameters. It is this variety of parameters to consider,which make pharmaceutical formulations a complex technical discipline.

For instance, and as example, a drug utilized for the preparation ofpharmaceutical formulations should meet an acceptable purity. There areestablished guidelines that define the limits and qualification ofimpurities in new drug substances produced by chemical synthesis, i.e.actual and potential impurities most likely to arise during thesynthesis, purification, and storage of the new drug substance.Guidelines are instituted for the amount of allowed degradation productsof the drug substance, or reaction products of the drug substance withan excipient and/or immediate container/closure system.

Stability is also a parameter considered in creating pharmaceuticalformulations. A good stability will ensure that the desired chemicalintegrity of drug substances is maintained during the shelf-life of thepharmaceutical formulation, which is the time frame over which a productcan be relied upon to retain its quality characteristics when storedunder expected or directed storage conditions. During this period thedrug may be administered with little or no risk, as the presence ofpotentially dangerous degradation products does not pose prejudicialconsequences to the health of the receiver, nor the lower content of theactive ingredient could cause under-medication.

Different factors, such as light radiation, temperature, oxygen,humidity, pH sensitivity in solutions, may influence stability and maydetermine shelf-life and storage conditions.

Bioavailability is also a parameter to consider in drug delivery designof pharmaceutically acceptable formulations. Bioavailability isconcerned with the quantity and rate at which the intact form of aparticular drug appears in the systemic circulation followingadministration of the drug. The bioavailability exhibited by a drug isthus of relevance in determining whether a therapeutically effectiveconcentration is achieved at the site(s) of action of the drug.

Physico-chemical factors and the pharmaco-technical formulation can haverepercussions in the bioavailability of the drug. As such, severalproperties of the drug such as dissociation constant, dissolution rate,solubility, polymorphic form, particle size, are to be considered whenimproving the bioavailability.

It is also relevant to establish that the selected pharmaceuticalformulation is capable of manufacture, more suitably, of large-scalemanufacture.

In view of the various and many technical requirements, and itsinfluencing parameters, it is not obvious to foresee whichpharmaceutical formulations will be acceptable. As such, it wasunexpectedly found that certain modifications of the solid state ofcompound of formula (X) positively influenced its applicability inpharmaceutical formulations.

SUMMARY OF THE INVENTION

Present invention concerns pseudopolymorphic forms of compound offormula (X) for the preparation of pharmaceutical formulations. Suchpseudopolymorphic forms contribute to pharmaceutical formulations inimproved stability and bioavailability. They can be manufactured insufficient high purity to be acceptable for pharmaceutical use, moreparticularly in the manufacture of a medicament for inhibiting HIVprotease activity in mammals.

In a first aspect, the present invention provides pseudopolymorphs of(3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl(1S,2R)-3-[[(4-aminophenyl)sulfonyl](isobutyl)amino]-1-benzyl-2-hydroxypropylcarbamate.

Pseudopolymorphs provided include alcohol solvates, more in particular,C1-C4 alcohol solvates; hydrate solvates; alkane solvates, more inparticular, C1-C4 chloroalkane solvates; ketone solvates, more inparticular, C1-C5 ketone solvates; ether solvates, more in particular,C1-C4 ether solvates; cycloether solvates; ester solvates, more inparticular, C1-C5 ester solvates; and sulfonic solvates, more inparticular, C1-4 sulfonic solvates, of the compound of formula (X).Preferred pseudopolymorphs are pharmaceutically acceptable solvates,such as hydrate and ethanolate.

Particular pseudopolymorphs are Form A (ethanolate), Form B (hydrate),Form C (methanolate), Form D (acetonate), Form E (dichloromethanate),Form F (ethylacetate solvate), Form G (1-methoxy-2-propanolate), Form H(anisolate), Form I (tetrahydrofuranate), Form J (isopropanolate) ofcompound of formula (X). Another particular pseudopolymorph is Form K(mesylate) of compound of formula (X).

In a second aspect, present invention relates to processes for preparingpseudopolymorphs. Pseudopolymorphs of compound of formula (X) areprepared by combining compound of formula (X) with an organic solvent,water, or mixtures of water and water miscible organic solvents, andapplying any suitable technique to induce crystallization, to obtain thedesired pseudopolymorphs.

In a third aspect, the invention relates to the use of the presentpseudopolymorphs, in the manufacture of pharmaceutical foiutulations forinhibiting HIV protease activity in mammals. In relation to thetherapeutic field, a preferred embodiment of this invention relates tothe use of pharmaceutically acceptable pseudopolymorphic forms ofcompound of formula (X) for the treatment of an HIV viral disease in amammal in need thereof, which method comprises administering to saidmammal an effective amount of a pharmaceutically acceptablepseudopolymorphic form of compound of formula (X).

The following drawings provide additional information on thecharacteristics of the pseudopolymorphs according to present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1, FIG. 2 and FIG. 3 are the powder X-ray diffraction patterns ofthe Form A (1:1).

FIG. 4 depicts Form A (1:1) in three dimensions with the atomsidentified.

FIG. 5 is a comparison of the Raman spectra of Forms A, B, D, E, F, H,(1:1) and the amorphous form at the carbonyl stretching region of1800-100 cm⁻¹ and the region 3300-2000 cm⁻¹.

FIG. 6 is a comparison of the expanded Raman spectra of Forms A, B, D,E, F, H, (1:1) and the amorphous form at the carbonyl stretching regionof 600-0 cm⁻¹.

FIG. 7 is a comparison of the expanded Raman spectra of Forms A, B, D,E, F, H, (1:1) and the amorphous form at the carbonyl stretching regionof 1400-800 cm⁻¹. In FIGS. 5, 6, and 7, P1 corresponds to Form A, P18corresponds to Form B, P19 corresponds to amorphous form, P25corresponds to Form E, P27 corresponds to Form F, P50 corresponds toForm D, P68 corresponds to Form H, P69 corresponds to Form C, P72corresponds to Form I, and P81 corresponds to Foul). G.

FIG. 8 is the Differential Scanning calorimetric (DSC) thermograph ofForm A (1:1).

FIG. 9 is the Infrared (IR) spectrum that reflects the vibrational modesof the molecular structure of Form A as a crystalline product

FIG. 10 is the IR spectrum that reflects the vibrational modes of themolecular structure of Form B as a crystalline product

FIG. 11: IR spectrum of forms A, B, and amorphous form, at spectralrange 4000 to 400 cm⁻¹.

FIG. 12: IR spectrum of forms A, B, and amorphous form, at spectralrange 3750 to 2650 cm⁻¹.

FIG. 13: IR spectrum of forms A, 13, and amorphous form, at spectralrange 1760 to 1580 cm⁻¹.

FIG. 14: IR spectrum of forms A, B, and amorphous form, at spectralrange 980 to 720 cm⁻¹. In FIGS. 11, 12, 13 and 14, curve A correspondsto Form A, curve B corresponds to Form 13, and curve C corresponds tothe amorphous form.

FIG. 15: DSC Thermograph curves of Form A (curve D), Form A afterAdsorption/Desorption (ADS/DES) (curve E), and Form A after ADS/DEShydration tests (curve F)

FIG. 16: Thermogravimetric (TG) curves of Form A (curve D), Form A afterADS/DES (curve E), and Form A after ADS/DES hydration tests (curve F)

FIG. 17: TG curve of Form A at 25° C. under dry nitrogen atmosphere infunction of time

FIG. 18: ADS/DES curves of Form A.

FIG. 19: ADS/DES curves of the hydration test of Form A

FIG. 20: ADS/DES curves of Form B

FIG. 21: IR spectrum of Form K

FIG. 22: Raman spectrum of Form K

FIG. 23: DSC curve of Form K

FIG. 24: TG curve of Form K

FIG. 25: ADS/DES isotherm of Form K, batch

FIG. 26: ADS/DES isotherm of Form K, batch 2

DETAILED DESCRIPTION

The term “polymorphism” refers to the capacity of a chemical structureto occur in different forms and is known to occur in many organiccompounds including drugs. As such, “polymorphic forms” or “polymorphs”include drug substances that appear in amorphous form, in crystallineform, in anhydrous form, at various degrees of hydration or solvation,with entrapped solvent molecules, as well as substances varying incrystal hardness, shape and size. The different polymorphs vary inphysical properties such as solubility, dissolution, solid-statestability as well as processing behaviour in terms of powder flow andcompaction during tabletting.

The term “amorphous form” is defined as a form in which athree-dimensional long-range order does not exist. In the amorphous formthe position of the molecules relative to one another are essentiallyrandom, i.e. without regular arrangement of the molecules on a latticestructure.

The term “crystalline” is defined as a form in which the position of themolecules relative to one another is organised according to athree-dimensional lattice structure.

The term “anhydrous form” refers to a particular form essentially freeof water. “Hydration” refers to the process of adding water molecules toa substance that occurs in a particular form and “hydrates” aresubstances that are formed by adding water molecules. “Solvating” refersto the process of incorporating molecules of a solvent into a substanceoccurring in a crystalline form. Therefore, the term “solvate” isdefined as a crystal form that contains either stoichiometric ornon-stoichiometric amounts of solvent. Since water is a solvent,solvates also include hydrates. The term “pseudopolymorph” is applied topolymorphic crystalline forms that have solvent molecules incorporatedin their lattice structures. The term pseudopolymorphism is usedfrequently to designate solvates (Byrn, Pfeiffer, Stowell, (1999)Solid-state Chemistry of Drugs, 2nd Ed., published by SSCI, Inc).

The present invention provides pseudopolymorphs of(3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl(1S,2R)-3-[[(4-aminophenyl)sulfonyl](isobutyl)amino]-1-benzyl-2-hydroxypropylcarbamate.

In one embodiment pseudopolymorphs are alcohol solvates, more inparticular, C₁-C₄ alcohol solvates; hydrate solvates; alkane solvates,more in particular, C₁-C₄ chloroalkane solvates; ketone solvates, morein particular, C₁-C₅ ketone solvates; ether solvates, more in particularC₁-C₄ ether solvates; cycloether solvates; ester solvates, more inparticular C₁-C₅ ester solvates; or sulfonic solvates, more inparticular, C₁-C₄ sulfonic solvates, of the compound of formula (X). Theterm “C₁-C₄ alcohol” defines straight and/or branched chained saturatedand unsaturated hydrocarbons having from 1 to 4 carbon atoms substitutedwith at least a hydroxyl group, and optionally substituted with analkyloxy group, such as, for example, methanol, ethanol, isopropanol,butanol, 1-methoxy-2-propanol and the like. The term “C₁-C₄chloroalkane” defines straight and/or branched chained saturated andunsaturated hydrocarbons having from 1 to 4 carbon atoms substitutedwith at least one chloro atom, such as, for example, dichloromethane.The term “C₁-C₅ ketone” defines solvents of the general formulaR′—C(═O)—R wherein R and R′ can be the same or different and are methylor ethyl, such as, acetone and the like. The term “C₁-C₄ ether” definessolvents of the general formula R′—O—R wherein R and R′ can be the sameor different and are a phenyl group, methyl or ethyl, such as, anisoleand the like. The term “cycloether” defines a 4- to 6-memberedmonocyclic hydrocarbons containing one or two oxygen ring atoms, such astetrahydrofuran and the like. The term “C₁-C₅ ester” defines solvents ofthe general formula R′—O—C(═O)—R wherein R and R′ can be the same ordifferent and are methyl or ethyl, such as ethylacetate and the like.The term “C₁-C₄ sulfonic solvent” defines solvents of the generalformula R—SO₃H wherein R can be a straight or branched chained saturatedhydrocarbon having from 1 to 4 carbon atoms, such as mesylate,ethanesulfonate, butanesulfonate, 2-methyl-1-propanesulfonate, and thelike.

Pseudopolymorphs of the present invention, which are pharmaceuticallyacceptable, for instance hydrates, alcohol solvates, such as,ethanolate, are preferred forms.

Several pseudopolymorphs are exemplified in this application and includeForm A (ethanolate), Form B (hydrate), Form C (methanolate), Form D(acetonate), Form E (dichloromethanate), Form F (ethylacetate solvate),Form G (1-methoxy-2-propanolate), Form H (anisolate), Form I(tetrahydrofuranate), Form J (isopropanolate), or Form K (mesylate) ofcompound of formula (X).

Solvates can occur in different ratios of solvation. Solvent content ofthe crystal may vary in different ratios depending on the conditionsapplied. Solvate crystal forms of compound of formula (X) may compriseup to 5 molecules of solvent per molecule of compound of formula (X),appearing in different solvated states including, amongst others,hemisolvate, monosolvate, disolvate, trisolvate crystals, intermediatesolvates crystals, and mixtures thereof. Conveniently, the ratio ofcompound of formula (X) to the solvent may range between (5:1) and(1:5). In particular, the ratio may range from about 0.2 to about 3molecules of solvent per 1 molecule of compound of formula (X), more inparticular, the ratio may range from about 1 to about 2 molecules ofsolvent per 1 molecule of compound of formula (X), preferably the ratiois 1 molecule of solvent per 1 molecule of compound of formula (X).

Solvates may also occur at different levels of hydration. As such,solvate crystal forms of compound of formula (X) may in additioncomprise under certain circumstances, water molecules partially or fullyin the crystal structures. Consequently, the term “Form A” will be usedherein to refer to the ethanolate forms of compound of formula (X)comprising up to 5 molecules of solvent per 1 molecule of compound offormula (X), intermediate solvates crystals, and the mixtures thereof;and optionally comprising additional water molecules, partially or fullyin the crystal structures. The same applies for Form B through Form K.In case a particular “Form A” needs to be denoted, the ratio ofsolvation will follow the “Form A”, for instance, one molecule ofethanol per one molecule of compound (X) is denoted as Form A (1:1).

The X-ray powder diffraction is a technique to characterise polymorphicforms including pseudopolymorphs of compound of formula (X) and todifferentiate solvate crystal forms from other crystal and non-crystalforms of compound of formula (X). As such, X-ray powder diffractionspectra were collected on a Phillips PW 1050/80 powder diffractometer,model Bragg-Brentano. Powders of Form A (1:1), around 200 mg eachsample, were packed in 0.5 mm glass capillary tubes and were analysedaccording to a standard method in the art. The X-ray generator wasoperated at 45 Kv and 32 mA, using the copper Ka line as the radiationsource. There was no rotation of the sample along the chi axis and datawas collected between 4 and 60° 2-theta step size. Form A (1:1) has thecharacteristic two-theta angle positions of peaks as shown in FIGS. 1, 2and 3 at: 7.04°±0.5°, 9.24°±0.5°, 9.96°±0.5′, 10.66°±0.5′, 11.30°±0.5°,12.82°±0.5°, 13.80°±0.5°, 14.56°±0.5°, 16.66°±0.5°, 17.30°±0.5°,18.28°±0.5°, 19.10°±0.5°, 20.00°±0.5°, 20.50° 0.5′, 21.22°±0.5°,22.68°±0.5°, 23.08°±0.5°, 23.66°±0.5°, 25.08°±0.5°, 25.58°±0.5°,26.28°±0.5°, 27.18°±0.5°, 28.22°±0.5°, 30.20°±0.5′, 31.34°±0.5°, 32.68°0.5′, 33.82°±0.5′, 39.18°±0.5°, 41.20°±0.5°, 42.06°±0.5°, and48.74°±0.5°.

In another set of analytical experiments, X-ray single diffraction wasapplied to Form A (1:1), which resulted in the following crystalconfiguration, listed in the table below.

TABLE 1 Crystal Data Crystal shape Prism Crystal dimensions 0.56 × 0.38× 0.24 mm Crystal color Colorless Space Group P 2₁ 2₁ 2₁ orthorhombicTemperature 293 K Cell constants a = 9.9882(6) Å b = 16.1697(8) Å c =19.0284(9) Å alpha (α) = 90° beta (β) = 90° gamma (γ) = 90° Volume3158.7(3) Å³ Molecules/unit cell (Z) 4 Density, in Mg/m³ 1.248 μ (linearabsorption coefficient) 1.340 mm⁻¹ F(000) 1272 Intensity MeasurementsDiffractometer Siemens P4 Radiation Cu Kα (λ = 1.54184 {acute over (Å)})Temperature ambient 2θ_(max) 138.14° Correction Empirical via Ψ-scansNumber of Reflections Measured Total: 3912 Structure Solution andRefinement Number of Observations 3467 [F² > 2 σ(F²)] Residual (R)0.0446

The resulting three-dimensional structure of Form A (1:1) is depicted inFIG. 4.

Table 2 shows the atomic coordinates (×10⁴) and equivalent isotropicdisplacement parameters (Å²×10³) for Form A (1:1). Atoms are numbered asexhibited in FIG. 4. The x, y and z fractional coordinates indicate theposition of atoms relative to the origin of the unit cell. U(eq) isdefined as one third of the trace of the orthogonalized tensor.

x y z U(eq) O1 7778(3) 2944(2) 9946(1) 70(1) C2 7171(4) 3513(2) 9487(2)64(1) C3 6831(3) 3046(2) 8823(2) 52(1) C3A 7953(3) 2411(2) 8793(2) 55(1)C4 7527(4) 1533(2) 8708(2) 65(1) C5 7425(5) 1241(2) 9457(2) 70(1) O68501(3) 1642(2) 9809(1) 76(1) C6A 8582(4) 2416(2) 9534(2) 62(1) O75533(2) 2702(1) 8945(1) 51(1) O8 5168(2) 2636(1) 7768(1) 53(1) C94791(3) 2534(1) 8368(1) 42(1) N10 3590(2) 2256(1) 8562(1) 43(1) C112638(3) 1916(2) 8068(2) 44(1) C12 2223(3) 1071(2) 8310(2) 58(1) C133381(3)  501(2) 8387(2) 56(1) C14 3937(4)  340(2) 9038(2) 67(1) C154989(5) −200(2) 9111(3) 80(1) C16 5494(5) −581(3) 8530(3) 96(2) C174975(6) −413(3) 7881(3) 98(2) C18 3926(5)  126(2) 7810(2) 78(1) C191423(3) 2464(2) 7976(2) 45(1) O20  494(2) 2112(1) 7502(1) 61(1) C211829(3) 3307(2) 7740(2) 48(1) N22  699(3) 3880(1) 7721(1) 49(1) C23 521(4) 4312(2) 7048(2) 58(1) C24  −61(4) 3785(2) 6473(2) 67(1) C25−1453(5)  3497(3) 6654(2) 86(2) C26  −47(7) 4247(3) 5779(2) 102(2)  S27 510(1) 4414(1) 8440(1) 50(1) O28  572(3) 3860(1) 9015(1) 61(1) O29−693(2) 4873(1) 8345(1) 65(1) C30 1854(3) 5080(2) 8509(2) 50(1) C311803(3) 5825(2) 8159(2) 54(1) C32 2871(4) 6341(2) 8195(2) 56(1) C334033(4) 6133(2) 8564(2) 55(1) C34 4063(4) 5385(2) 8909(2) 59(1) C352998(4) 4869(2) 8883(2) 56(1) N36 5076(3) 6667(2) 8596(2) 72(1) C37 1920(10) 2231(7) 5258(4) 232(6)  C38  1310(10) 1590(6) 5564(4) 191(5) O39 1768(4) 1393(2) 6249(2) 94(1)

Table 3 shows the anisotropic displacement parameters (Å²×10³) for FormA (1:1). The anisotropic displacement factor exponent takes the formula:

−2π² [h ² a* ² U ₁₁+ . . . +2hka*b*U ₁₂]

U₁₁ U₂₂ U₃₃ U₂₃ U₁₃ U₁₂ O1 65(2) 89(2) 55(1) −4(1) −12(1) −3(1) C2 53(2)68(2) 71(2) −7(2) −8(2) −11(2)  C3 38(2) 63(2) 55(2) 4(1) −2(1) −12(1) C3A 37(2) 78(2) 49(1) 9(1) 1(1) −3(2) C4 61(2) 74(2) 61(2) −4(2) −6(2)10(2) C5 72(3) 67(2) 71(2) 8(2) −11(2) −7(2) O6 78(2) 80(2) 70(1) 16(1)−21(1) −8(2) C6A 47(2) 80(2) 59(2) 5(2) −6(2) −7(2) O7 34(1) 69(1) 50(1)0(1) −1(1) −9(1) O8 42(1) 68(1) 50(1) 3(1) 2(1) −12(1)  C9 35(2) 41(1)49(1) 1(1) −3(1)  3(1) N10 31(1) 50(1) 49(1) −1(1) 1(1) −2(1) C11 32(2)41(1) 57(1) −4(1) 0(1) −2(1) C12 44(2) 42(1) 87(2) 2(1) 2(2) −4(1) C1350(2) 39(1) 78(2) 0(1) 8(2)  0(1) C14 64(2) 56(2) 80(2) 0(2) 5(2)  9(2)C15 68(3) 72(2) 100(3) 18(2) 7(2) 12(2) C16 77(3) 68(2) 143(4) 26(3)34(3) 28(2) C17 114(4)  72(2) 109(3) −6(2) 32(3) 38(3) C18 89(3) 60(2)85(2) −4(2) 10(2) 10(2) C19 30(2) 44(1) 61(1) −3(1) −5(1) −5(1) O2044(1) 56(1) 83(1) −6(1) −18(1) −6(1) C21 36(2) 42(1) 64(2) 2(1) −4(1)−1(1) N22 42(1) 47(1) 57(1) 1(1) 0(1)  3(1) C23 59(2) 50(1) 64(2) 7(1)−8(2)  1(2) C24 79(3) 59(2) 62(2) 1(1) −11(2)  6(2) C25 75(3) 83(2)101(3) 6(2) −30(3) −5(2) C26 143(5)  99(3) 65(2) 14(2) −15(3) −6(3) S2744(1) 47(1) 61(1) 2(1) 2(1)  1(1) O28 64(2) 58(1) 61(1) 9(1) 3(1) −7(1)O29 46(1) 58(1) 92(2) −4(1) 6(1) 10(1) C30 50(2) 46(1) 54(1) 2(1) 1(1) 1(1) C31 50(2) 48(1) 64(2) 6(1) −4(2)  6(1) C32 59(2) 45(1) 65(2) 4(1)2(2)  1(1) C33 57(2) 55(2) 52(1) −4(1) 1(1) −3(1) C34 56(2) 63(2) 59(2)6(1) −13(2) −3(2) C35 63(2) 52(1) 53(1) 5(1) −8(2) −2(2) N36 67(2) 70(2)80(2) 4(2) −5(2) −19(2)  C37 290(10) 260(10) 145(7) 68(7) 67(8) 120(10)C38 280(10) 187(7)  104(4) 1(5) −53(6) −80(10) O39 99(2) 91(2) 93(2)1(2) −13(2) −28(2) 

Raman spectroscopy has been widely used to elucidate molecularstructures, crystallinity and polymorphism. The low-frequency Ramanmodes are particularly useful in distinguishing different molecularpackings in crystal. As such, Raman spectra were recorded on a BrukerFT-Raman RFS 100 spectrometer equipped with a photomultiplier tube andoptical multichannel detectors. Samples placed in quartz capillary tubeswere excited by an argon ion laser. The laser power at the samples wasadjusted to about 100 mW and the spectral resolution was about 2 cm⁻¹.It was found that Forms A, B, D, E, F, and H, (1:1) and the amorphousform have the Raman spectra which appear in FIGS. 5, 6, and 7.

In addition, Forms A and B were characterized using a μATR(Micro-Attenuated Total Reflectance) accessory (Harrick Split-Pea withSi crystal). The infrared spectra were obtained with a Nicolet Magna 560FTIR spectrophotometer, a Ge on KBr beamsplitter, and a DTGS with KBrwindows detector. Spectra were measured at 1 cm⁻¹ resolution and 32scans each, in a wavelength range of from 4000 to 400 cm⁻¹, andapplication of baseline correction. The wavenumbers for Form A obtainedare exhibited in the following Table 4.

TABLE 4 Wavenumbers (cm⁻¹) and relative intensities of absorption bands(¹) 3454w, 3429w, 3354w, 3301w, 3255w, 3089w, 3060w, 3041w, 3028w 2964w,2905w, 2875w, 2856w, 2722vw, 2684vw, 2644vw, 2603vw, 2234vw 1704s,1646w, 1595s, 1550m, 1503m, 1466w, 1453w, 1444w, 1413w 1373w, 1367w,1340w, 1324m, 1314m, 1306m, 1290w, 1266m, 1244m, 1229m 1187w, 1146s,1124m, 1104m, 1090m, 1076m, 1052m, 1042s, 1038m, 1024s 987s, 971m, 944m,909w, 890w, 876w, 841m, 792w, 768s, 742s, 732w, 697m, 674s, 645w, 630m598w, 593w, 574m, 564s, 553vs, 538m, 533m, 531m, 526m, 508m, 501m, 491m,471m, 458w, 445w, 442w, 436w, 428w, 418w vs = very strong, s = strong, m= medium, w = weak, vw = very weak, br = broad

The IR spectrum in FIG. 9 reflects the vibrational modes of themolecular structure as a crystalline product.

The wavenumbers obtained for Form B are exhibited in the following Table5.

TABLE 5 Wavenumbers (cm⁻¹) and relative intensities of absorption bands(¹) 3614w, 3361m, 3291m, 3088w, 3061w, 3043w, 3028w 2967w, 2905w, 2872w,2222vw 1703s, 1631w, 1595s, 1553m, 1502w, 1467w, 1453w, 1444w, 1436w1388vw, 1374vw, 1366w, 1355vw, 1340w, 1308m, 1291w, 1267m, 1245m 1187w,1148s, 1125m, 1105m, 1091m, 1077m, 1052m, 1044m, 1025s 990m, 972w, 944m,912w, 891w, 876vw, 862w, 843w, 836w, 792w, 769m, 757w, 743m, 717w, 699m,672m 598w, 591w, 585w, 576m, 566m, 553vs, 536m, 509w, 502m, 484w, 471w,432vw, 425w, 418w (¹) vs = very strong, s = strong, m = medium, w =weak, vw = very weak, br = broad

The IR spectrum in FIG. 10 reflects the vibrational modes of themolecular structure of Form B as a crystalline product.

Following the same analytical IR method, Form B and the amorphous formwere also characterised and compared with Form A, as shown in FIGS. 11to 14. IR spectra of the different physical forms showed distinctspectral differences, most relevant are those in Table 6:

TABLE 6 Wavenumbers (cm⁻¹) and relative intensities of absorption bands(¹) Form A Form B Amorphous form 3454m, 3429m, 3353m, 3615m, 3356m,3291m, 3462m, 3362m, 3255m, 3089w, 3060m, 3089m, 3061m, 3043w, 3249m,3062m, 3041w, 3028w 3027w 3026m 2963m, 2905m, 2869m, 2966m, 2905m, 2873m2959m, 2871m 2856m 1704s, 1646m, 1596s, 1703s, 1630m, 1595s, 1704s,1628s, 1549s, 1503s 1552s, 1502m 1596s, 1525s, 1502s 1306s, 1266s, 1244s1308s, 1267s, 1245s 1312s, 1259s 1146s, 1104s, 1090s, 1148s, 1105s,1090s, 1143s, 1090s, 1076s, 1052s, 1042s, 1077s, 1052s, 1044s, 1014s1038s, 1023s 1024s 987s, 971s, 954s, 945s, 989s, 972s, 944s, 925m, 960s,953s, 950s, 912m, 909m, 891s, 876s, 915m, 912s, 891s, 862s, 944s, 937s,922s, 841s, 827s 843s 832s 792m, 768s, 742s, 697s, 792s, 769s, 744s,699s, 750br, 702s, 672s 674s 672s (¹) s = strong, m = medium, w = weak,vw = very weak, br = broad

The physical Forms A, B, and amorphous form are identified throughspectral interpretation, focused on absorption bands specific for eachform. Unique and specific spectral differences between forms are noticedin 3 spectral ranges: from 3750 to 2650 cm⁻¹ (range 1), from 1760 to1580 cm⁻¹ (range 2) and from 980 to 720 cm⁻¹ (range 3).

Range 1 (from 3750 to 2650 cm⁻¹)

FIG. 11: Form A shows a double band with absorption maxima at 3454 cm⁻¹and 3429 cm⁻¹. Form B shows a single absorption band at 3615 cm⁻¹ andamorphous form shows a single absorption band at 3362 cm⁻¹.

Range 2 (from 1760 to 1580 cm⁻¹)

FIG. 12: Form A shows a single absorption band at 1646 cm⁻¹, Form Bshows a single absorption band at 1630 cm⁻¹ and amorphous form shows asingle absorption band at 1628 cm⁻¹ with a clearly higher intensitycompared to the Form B band. Additionally, amorphous form shows a lessintense, broad band at 1704 cm⁻¹ compared to both forms A and B bands atabout 1704 cm⁻¹.

Range 3 (from 980 to 720 cm⁻¹)

FIG. 13: Form A shows a distinct set of 5 absorption bands at 911, 890,876, 862 and 841 cm⁻¹. Form B shows a similar set but the 876 cm⁻¹ bandis missing. Amorphous form shows a single broad band at about 750 cm⁻¹,both forms A and B show two maxima at about 768 cm⁻¹ and 743 cm⁻¹.

Thermomicroscopy is another useful technique in the study of solid-statekinetics. The kinetics of nucleation processes from solutions or melts,including the analysis of the nucleation speed, can be quantified. Thesimplest and most widely used method is the melting point determination.As such, a Mettler model FP 82 controller with heating stage was used ona Leitz microscope. A few particles of Form A were placed on a glassslide and observed while heating at 10° C. per minute. The melting rangefor Form A (1:1) was found to be between 90° and 110° C.

On another means of characterization, the solubility of Form A (1:1) wasalso a matter subject to study. Its solubility in different solvents atapproximate 23° C. was determined to be as follows:

TABLE 7 Approximate solubility for Form A (1:1), in mg/ml Approximatesolubility Solvent Form A (mg/ml) Acetone 106-211 Dichloromethane105-209 1-Methoxy-2-propanol 160-213 Ethylmethylketone 102-204Ethylacetate  71-107 Ethanol absolute <3.4 Heptane <3.4 Water <3.5Isopropylether <3.4 Methacyanate >200 Methanol <3.4 2-Propanol <3.4Tetrahydrofurane 102-203 Toluene <3.5

Further solubility investigations were performed in function of pH. Assuch, the aqueous solubilities of Form. A (1:1) were measured insolvents with different pH. An excess of the solute was equilibratedwith the solvent at 20° C. for at least 24 hours. After removing theundissolved compound, the concentration in solution was determined usingUV spectrometry.

TABLE 8 Solubility for Form A (1:1) in function of pH Solvent Solubility(mg/100 ml solution) Water 16 (pH 5.9) Buffer pH 2 (citrate/HCl) 18 (pH2.0) Buffer pH 3 (citrate/HCl) 10 (pH 3.0) Buffer pH 4 (citrate/HCl) 9(pH 4.0) 0.01N HCl 18 (pH 2.1) 0.1N HCl 83 (pH 1.1) 1.0N HCl 620 (pH0.2)

Solubility of Form A (1:1) in function of HPPCD(hydroxypropyl-β-cyclodextrin) was measured. An excess of product wasequilibrated with the solvent during 2 days at 20° C. After removing theundissolved compound, the concentration in solution was determined usingUV spectrometry.

TABLE 9 Solubility for Form A (1:1) in function of HPβCD solventSolubility in mg/ml solution Water 0.16 (pH = 5.9) 5% HPβCD in water 2.4(pH = 5.8) 10% HPβCD in water 6.5 (pH = 6.0) 20% HPβCD in water 17 (pH =6.0) 40% HPβCD in water 40 (pH = 5.9)

In a second aspect, the present invention relates to processes forpreparing pseudopolymorphs. Pseudopolymorphs of compound of formula (X)are prepared by combining compound of formula (X) with an organicsolvent, or water, or mixtures of water and water miscible organicsolvents, applying any suitable technique to induce crystallization, andisolating the desired pseudopolymorphs.

By techniques for inducing crystallization are to be understood thoseprocesses for the production of crystals, which include amongst others,dissolving or dispersing compound of formula (X) in a solvent medium,bringing the solution or dispersion of compound of formula (X) and thesolvent(s) to a desired concentration, bringing the said solution ordispersion to a desired temperature, effecting any suitable pressure,removing and/or separating any undesired material or impurities, dryingthe formed crystals to obtain the pseudopolymorphs in a solid state, ifsuch state is desired.

Bringing the solution or dispersion of compound of formula (X) andsolvents to a desired concentration does not necessarily imply anincrease in the concentration of compound of formula (X). In certaincases, a decrease or no change in concentration could be preferable. Bybringing the said solution or dispersion to a desired temperature, onewill understand the acts of heating, cooling or leaving at ambienttemperature.

The techniques used for obtaining a desired concentration are thosecommon in the art, for instance, evaporation by atmosphericdistillation, vacuum distillation, fractioned distillation, azeotropicdistillation, film evaporation, other techniques well known in the artand combinations thereof. An optional process for obtaining a desiredconcentration could as well involve the saturation, of the solution ofcompound of formula (X) and solvent, for example, by adding a sufficientvolume of a non-solvent to the solution to reach the saturation point.Other suitable techniques for saturating the solution include, by way ofexample, the introduction of additional compound of formula (X) to thesolution and/or evaporation of a portion of the solvent from thesolution. As referred to herein, saturated solution encompassessolutions at their saturation points or exceeding their saturationpoints, i.e. supersaturated.

Removing and/or separating any undesired material or impurities may beperformed by purification, filtering, washing, precipitation or similartechniques. Separation, for example, can be conducted by knownsolid-liquid separation techniques. Filtering procedures known to thoseskilled in the art can as well be used in the present process. Thefiltrations can be performed, amongst other methods, by centrifugation,or using Buchner style filter, Rosenmund filter or plates, or framepress. Preferably, in-line filtration or safety filtration may beadvantageously intercalated in the processes disclosed above, in orderto increase the purity of the resulting pseudopolymorphic form.Additionally, filtering agents such as silica gel, Arbocel®, dicalitediatomite, or the like, may also be employed to separate impurities fromthe crystals of interest.

Crystals obtained may be also dried, and such drying process mayoptionally be used in the different crystallization passages, if morethan one crystallization passage is applied. Drying procedures includeall techniques known to those skilled in the art, such as heating,applying vacuum, circulating air or gas, adding a desiccant,freeze-drying, spray-drying, evaporating, or the like, or anycombination thereof.

Processes for crystallization of pseudopolymorphs of compound of formula(X) embrace multiple combinations of techniques and variations thereof.As such, and by way of example, crystallization of pseudopolymorphs ofcompound of formula (X) may be executed by dissolving or dispersingcompound of formula (X) at a suitable temperature in the solvent wherebyportion of the said solvent evaporates increasing the concentration ofthe compound of formula (X) in the said solution or dispersion, coolingthe said mixture, and optionally washing and/or filtering and dryingresulting solvate crystals of compound of formula (X). Optionally,pseudopolymorphs of compound of formula (X) may be prepared bydissolving or dispersing compound of formula (X) in a solvent medium,cooling said solution or dispersion and subsequently filtering anddrying the obtained pseudopolymorph. Another example of preparation ofsolvates of compound of formula (X) could be by saturating compound offormula (X) in the solvent medium, and optionally filtering, washing anddrying obtained crystals.

Crystal formation may as well involve more than one crystallizationprocess. In certain cases, one, two or more extra crystallization stepsmay be advantageously performed for different reasons, such as, toincrease the quality of the resulting solvate. For instance,pseudopolymorphs of the present invention could also be prepared byadding a solvent to an initial starting base material of compound offormula (X), stirring the solution at a fixed temperature until thesubstances would be fully solved, concentrating the solution by vacuumdistillation, and cooling. A first crystallization would take place andthe formed crystals would be newly washed with a solvent, and followedby dissolution of compound of formula (X) with the solvent to form thedesired pseudopolymorph. Recrystallization of the reaction mixture wouldoccur, followed by a cooling step from reflux. The formedpseudopolymorph would optionally be filtered and allowed to dry.

By dissolving or dispersing compound of formula (X) in the organicsolvent, water or a mixture of water and water miscible organicsolvents, one may obtain different degrees of dispersion, such assuspensions, emulsions, slurries or mixtures; or preferably obtainhomogeneous one-phase solutions.

Optionally, the solvent medium may contain additives, for example one ormore dispersing agents, surfactants or other additives, or mixturesthereof of the type normally used in the preparation of crystallinesuspensions and which are well documented in the literature. Theadditives may be advantageously used in modifying the shape of crystalby increasing the leniency and decreasing the surface area.

The solvent medium containing the solution may optionally be stirred fora certain period of time, or vigorously agitated using, for example, ahigh shear mixer or homogeniser or a combination of these, to generatethe desired droplet size for the organic compound.

Examples of organic solvents useful for the present invention includeC₁-C₄ alcohols such as methanol, ethanol, isopropanol, butanol,1-methoxy-2-propanol, and the like; C₁-C₄ chloroalkanes such asdichloromethane; C₁-C₄ ketones such as acetone; C₁-C₄ ethers such asanisole, and the like; cycloethers such as tetrahydrofuran; C₁-C₄ esterssuch as ethylacetate; sulfonates such as mesylate, ethanesulfonate,butanesulfonate, 2-methyl-1-propanesulfonate; and the like.

Examples of mixtures of water and water miscible organic solventsinclude, mixtures of water with all organic solvents listed aboveprovided they are miscible in water, e.g. ethanol/water, for instance ina 50/50 ratio.

Preferred solvents are those pharmaceutically acceptable solvents.However, pharmaceutically non-acceptable solvents may also find theiruse in the preparation of pharmaceutically acceptable pseudopolymorphs.

In a preferred method, the solvent is a pharmaceutically acceptablesolvent since it results in a pharmaceutically acceptablepseudopolymorph. In a more preferred method, the solvent is ethanol.

In a particular embodiment, pharmaceutically acceptable pseudopolymorphsof compound of formula (X) can be prepared starting frompseudopolymorphic forms of compound of formula (X), which may not benecessarily pharmaceutically acceptable. For instance, Form A may beprepared starting from Form J. Pseudopolymorphs may also be preparedstarting from the amorphous form.

In the mixtures of water and water miscible organic solvents, the amountof water can vary from about 5% by volume to about 95% by volume,preferably from about 25% to about 75% by volume, more preferably fromabout 40% to about 60% by volume.

It should also be noted that the quality of selected organic solvent(absolute, denaturated, or other) also influences the resulting qualityof the pseudopolymorph.

Control of precipitation temperature and seeding may be additionallyused to improve the reproducibility of the crystallization process, theparticle size distribution and form of the product. As such, thecrystallization can be effected without seeding with crystals of thecompound of the formula (X) or preferably in the presence of crystals ofthe compound of the formula (X), which are introduced into the solutionby seeding. Seeding can also be effected several times at varioustemperatures. The amount of the seed material depends on the amount ofthe solution and can readily be determined by a person skilled in theart.

The time for crystallization in each crystallization step will depend onthe conditions applied, the techniques employed and/or solvents used.

Breaking up the large particles or aggregates of particles after crystalconversion may additionally be performed in order to obtain a desiredand homogeneous particle size. Accordingly, the solvate crystal forms ofcompound of formula (X) are optionally milled after undergoingconversion. Milling or grinding refers to physically breaking up thelarge particles or aggregates of particles using methods and apparatuswell known in the art for particle size reduction of powders. Resultingparticle sizes may range from millimeters to nanometers, yielding i.e.nanocrystals, microcrystals.

The yield of the preparation process of the pseudopolymorphs of compoundof formula (X) may be 10% or more, a more preferred yield would varyfrom 40% to 100%.

Interestingly, the yield varies between 70% and 100%.

Suitably, pseudopolymorphs of the present invention have a puritygreater than 90 percent. More suitably, the present pseudopolymorphshave a purity greater than 95 percent. Even more suitably, the presentpseudopolymorphs have a purity greater than 99 percent.

In a third aspect, the present invention relates to a pharmaceuticalformulation comprising a therapeutically effective amount of apseudopolymorph of compound of formula (X), and a pharmaceuticallyacceptable carrier or diluent thereof.

In one embodiment, present invention relates to the use ofpharmaceutically acceptable pseudopolymorphic forms of compound offormula (X), preferably Form A, in the manufacture of a medicament fortreating diseases caused by retroviruses, such as HIV infections, forexample, Acquired Immune Deficiency Syndrome (AIDS) and AIDS-relatedcomplex (ARC).

In another embodiment, present invention provides a method for thetreatment of a retroviral infection, for example an HIV infection, in amammal such as a human, which comprises administering to the mammal inneed thereof an effective antiretroviral amount of a pharmaceuticallyacceptable pseudopolymorphic form of compound of formula (X), preferablyForm A.

Present invention also relates to a method in which the treatment of aHIV viral infection comprises the reduction of HIV load. Presentinvention also relates to a method in which the treatment of said HIVviral infection comprises the increase of CD4+ cell count. Presentinvention relates as well to a method in which the treatment of said HIVviral infection comprises inhibiting HIV protease activity in a mammal.

Pharmaceutically acceptable pseudopolymorphic forms of compound offormula (X), preferably Form A, also referred to herein as the activepharmaceutical ingredients, may be administered by any route appropriateto the condition to be treated, preferably orally. It will beappreciated however, that the preferred route may vary with, forexample, the condition of the recipient.

For each of the above-indicated utilities and indications the amountrequired of the active ingredient will depend upon a number of factorsincluding the severity of the condition to be treated and the identityof the recipient and will ultimately be at the discretion of theattendant physician or veterinarian. The desired dose preferably may bepresented as one, two, three or four or more subdoses administered atappropriate intervals throughout the day.

For an oral administration form, pseudopolymorphs of the presentinvention are mixed with suitable additives, such as excipients,stabilizers or inert diluents, and brought by means of the customarymethods into the suitable administration forms, such as tablets, coatedtablets, hard capsules, aqueous, alcoholic, or oily solutions. Examplesof suitable inert carriers are gum arabic, magnesia, magnesiumcarbonate, potassium phosphate, lactose, glucose, or starch, inparticular, corn starch. In this case the preparation can be carried outboth as dry and as moist granules. Suitable oily excipients or solventsare vegetable or animal oils, such as sunflower oil or cod liver oil.Suitable solvents for aqueous or alcoholic solutions are water, ethanol,sugar solutions, or mixtures thereof. Polyethylene glycols andpolypropylene glycols are also useful as further auxiliaries for otheradministration forms.

For subcutaneous or intravenous administration, the pseudopolymorphs ofcompound of formula (X), if desired with the substances customarytherefor such as solubilizers, emulsifiers or further auxiliaries, arebrought into solution, suspension, or emulsion. The pseudopolymorphs ofcompound of formula (X) can also be lyophilized and the lyophilizatesobtained used, for example, for the production of injection or infusionpreparations. Suitable solvents are, for example, water, physiologicalsaline solution or alcohols, e.g. ethanol, propanol, glycerol, inaddition also sugar solutions such as glucose or mannitol solutions, oralternatively mixtures of the various solvents mentioned.

Suitable pharmaceutical formulations for administration in the form ofaerosols or sprays are, for example, solutions, suspensions or emulsionsof the pseudopolymorphs of compound of formula (X) in a pharmaceuticallyacceptable solvent, such as ethanol or water, or a mixture of suchsolvents. If required, the formulation can also additionally containother pharmaceutical auxiliaries such as surfactants, emulsifiers andstabilizers as well as a propellant. Such a preparation customarilycontains the active compound in a concentration from approximately 0.1to 50%, in particular from approximately 0.3 to 3% by weight.

Pseudopolymorphs of the present invention may also be presented in aformulation comprising micrometer-, nanometer- or picometer-sizeparticles of the pseudopolymorph of compound of formula (X), whichformulation may contain other pharmaceutical agents and may optionallybe converted to solid form.

It may be convenient to formulate the present pseudopolymorphs in theform of nanoparticles which have a surface modifier adsorbed on thesurface thereof in an amount sufficient to maintain an effective averageparticle size of less than 1000 nm. Useful surface modifiers arebelieved to include those that physically adhere to the surface of theantiretroviral agent but do not chemically bind to the antiretroviralagent.

It may be further convenient to store the pseudopolymorphs of compoundof formula (X) in packaging materials which are protective tomechanical, environmental, biological or chemical hazards, ordegradation. Conditioning drug substances can be achieved by employingpackaging materials impermeable to moisture, such as sealed vapour lockbags. Conditioning drug products, such as tablets, capsules, can beachieved by employing for instance, aluminium blisters.

It should be understood that in addition to the ingredients particularlymentioned above, formulations of this invention includes other agentsconventional in the art having regard to the type of formulation inquestion, for example those suitable for oral administration may includeflavouring agents or taste masking agents.

The following examples are intended for illustration only and are notintended to limit the scope of the invention in any way.

Example 1

The industrial scale synthesis of Form A (1:1) was performed using thefollowing steps. First a solution was prepared with isopropanol and(3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl(1S,2R)-3-[[(4-aminophenyl)sulfonyl](isobutyl)amino]-1-benzyl-2-hydroxypropylcarbamate.The solution was concentrated by vacuum distillation at 70° C. and200-500 mbar pressure and cooled from a T>35° to a T between 15° and 20°C. for about 10 hours. The crystals formed were newly washed with 13liters isopropanol and filtered. A subsequent recrystallization fromethanol/water (90 liters/90 liters) was performed. This was followed bya new dissolution step, but with 60 liters ethanol instead.Recrystallization of the reaction mixture from ethanol occurred,followed by a cooling step from reflux to −15° C. approximately andduring 10 hours. The ethanolate formed was filtered and let to dry atabout 50° C. and about 7 mbar. The yield of this process was at least75%.

Example 2

In another example a mixture of Form D and Form B were prepared. Acetonewas used as a solvent during the crystallisation process to form Form D.The crystallisation process then comprised the step of stirring theinitial starting compound (10 g) in 70 ml acetone. The solution wassubsequently refluxed until the compound was completely solved. 40 ml ofwater were added and the solution was subsequently cooled slowly untilroom temperature and stirred overnight. Formed crystals were filteredand dried in the vacuum oven at 50° C. 7.6 g of product resulted fromthe crystallization, being the yield of this process of about 75%.

Example 3

In another example Form J crystals were prepared. Isopropanol was usedas a solvent during the crystallisation process to form Form J. Thecrystallisation process then comprised the step of solving the initialstarting material in the hot solvent. The solution was subsequentlycooled until room temperature. Formed crystals were filtered and driedin the vacuum oven at 50° C. The crystals contained about 50 mol %isopropanol.

Example 4

In this example, the mass losses for different pseudopolymorphs inthermogravimetric (TG) experiments were calculated. Thermogravimetry isa technique that measures the change in mass of a sample as it isheated, cooled or held at constant temperature. Approximately 2 to 5 mgof sample were placed on a pan and inserted into the TG furnace, modelNetzsch Thermo-Microbalance TG 209 coupled to a Bruker FTIR Spectrometervector 22. The samples were heated in a nitrogen atmosphere at a rate of10° C./min, up to a final temperature of 250° C. The detection limit ofresidual solvents was in the order of 0.1% for distinct stepwise solventloss over a narrow temperature range (few degrees Celsius).

The following TG data were obtained:

Form A: a weight loss of 4.2% was observed in the temperature range of25-138° C. (ethanol+little water) and of 6.9% (ethanol+CO₂) in thetemperature range of 25-200° C. Ethanol loss rate was maximal at 120° C.CO₂ loss was due to chemical degradation and was visible at around 190°C.

Form B: a weight loss of 3.4% was observed in the temperature range25-78° C. (water) and of 5.1% in the temperature range 25-110° C.(ethanol+water for T>78° C.). From 110-200° C. further 1.1% weight waslost (ethanol).

Form C: a weight loss of 2.1% was observed in the temperature range25-83° C. (water+methanol) and of 4.2% in the temperature range 25-105°C. (methanol for T>83° C., distinct step). From 105-200° C. further 2.1%weight was lost (methanol). No ethanol was observed in the gas phase.

Form D: a weight loss of 0.1% was observed in the temperature range25-50° C., of 4.2% in the temperature range 25-108° C. (acetone+ethanolfor T>50° C.), of 8.2% in the temperature range 25-157° C.(acetone+ethanol for T>108° C.) and of 10.5% in the temperature range25-240° C. (acetone+ethanol for T>157° C.).

Form E: a weight loss of 0.2% was observed in the temperature range25-75° C. (water), of 1.8% in the temperature range 25-108° C.(dichloromethane+ethanol for T>75° C.), of 6.8% in the temperature range25-157° C. (dichloromethane+ethanol for T>108° C.) and of 8.8% in thetemperature range 25-240° C. (dichloromethane-1-ethanol for T>157° C.).

Form F: a weight loss of 0.1% was observed in the temperature range25-50° C. (probably water), of 1.7% in the temperature range 25-108° C.(ethylacetate+ethanol for T>50° C.), of 6.6% in the temperature range25-157° C. (ethylacetate+ethanol for T>108° C.) and of 9% in thetemperature range 25-240° C. (ethylacetate+ethanol for T>157° C.).

Form G: a weight loss of 0.0% was observed in the temperature range25-50° C., of 3.7% in the temperature range 25-108° C.(1-methoxy-2-propanol+ethanol for T>50° C., distinct step), of 8% in thetemperature range 25-157° C. (1-methoxy-2-propanol+ethanol for T>108°C.) and of 12.5% in the temperature range 25-240° C.(1-methoxy-2-propanol+ethanol for T>157° C.).

Form H: a weight loss of 0.8% was observed in the temperature range25-100° C. (anisole+little ethanol) and of 8.8% in the temperature range25-200° C. (anisole ethanol for T>100° C.).

Form I: a weight loss of 0.3% was observed in the temperature range25-89° C. (water) and of 11.0% in the temperature range 25-200° C.(tetrahydrofurane for T>89° C.). No ethanol was observed in the gasphase.

Table 10 shows approximate expected mass losses for different Forms inthermogravimetric (TG) experiments.

Mass loss in % (M+x.LM 100%)

Pseudo- BP polymorph [° C.] Hemisolvate Monosolvate Disolvate TrisolvateForm D 56 5.0 9.6 17.5 24.1 Form H 152 9.0 16.5 28.3 37.2 Form E 40 7.213.4 23.7 31.8 Form G 119 7.6 14.1 24.8 33.1 Form F 76 7.4 13.9 24.332.6 Form A 78 4.0 7.8 14.4 20.2 Form B 100 1.6 3.2 6.2 9.0 Form C 652.8 5.5 10.5 14.9 Form I 66 6.2 11.6 20.8 28.3

In another set of thermogravimetric methods, Form A, Form A afterAdsorption/Desorption, and Form A after Adsorption/Desorption hydrationtests, were all transferred into an aluminum sample pan. The TG curvewas recorded on a TA Instrument Hi-Res TGA 2950 thermogravimeter at thefollowing conditions:

-   -   initial temperature: room temperature    -   heating rate: 20° C./min    -   resolution factor: 4    -   final condition: 300° C. or <80[(w/w) %]

The TG curves of the samples are collected in FIG. 16.

Table 11 shows mass losses for the forms tested:

TG (% weight change) Form A Up to 80° C. >80° C. Form A 0.3 7.1 Form Aafter ADS/DES 2.9 4.0 Form A after A/D hydratation test 5.4 0.5

The loss of weight at temperatures up to 80° C. is mainly due to theevaporation of solvent (water) present in the sample. The loss of weightat temperatures above 80° C. is mainly due to the evaporation of solvent(ethanolate) present in the sample.

A TG curve of form A at 25° C. under dry nitrogen atmosphere in functionof time is collected in FIG. 17. The loss of weight at 25° C. after 10hours was around 0.6%. This was due to the evaporation of solvent.

Example 5

In another example, measurements of differential scanning calorimetry(DSC) were also performed. For such purpose, a Perkin Elmer DSC 204thermal analysis system was used. From 2 to 5 mg sample of Form A wereaccurately weighed into a DSC pan.

The experiments were performed in an open pan. The sample wasequilibrated to approximately 30° C. and then heated at a rate of 10° C.per minute, up to a final temperature of 200° C. The DSC data wasobtained following a standard method in the art. The Fo A wascharacterized by differential scanning calorimetry (DSC) in which itshowed a sharp endotherm in the range 80-119° C., showing a peak atabout 105.6° C., with a delta H=−98.33 J/g onset. Accordingly, theethanol solvate crystal Form A of compound of formula (X) (1:1) showedthe thermograph pattern, which appears in FIG. 8.

In another set of DSC measurements, Form A, Form A afterAdsorptionlDesorption, and Form A after AdsorptionlDesorption hydrationtests were examined. About 3 mg of the samples were transferred into a30 μl perforated aluminum Perkin Elmer sample pan. The sample pan wasclosed with the appropriate cover and the DSC curve recorded on a PerkinElmer Pyris DSC, at the following conditions:

-   -   initial temperature: 25° C.    -   heating rate: 10° C./min    -   final temperature: 150° C.    -   nitrogen flow: 30 ml/min

Form A showed an endothermic signal at about 104.6° C. and a heat offusion of 95.8 J/g caused by the evaporation of the ethanolate and themelting of the product. Form A after ADS/DES showed a broad endothermicsignal due to a mixture of ethanolate Form A and hydrated Form B. Form Aafter ADS/DES hydration test showed an endothermic signal at about 73.5°C. and a heat of fusion of 126 J/g caused by the evaporation of waterand the melting of the product. Thermograph curves are depicted in FIG.15.

Example 6

In another example stability studies of the Form A in three differentconditions were tested out. They included conditions of 25° C. and 60%RH, 40° C. and 75% RH, and 50° C. These studies revealed that at 25° C.and 60% RH long-term stability, the amount of ethanol and water isstable.

Table 12 shows the Stability study for Form A. Long term stability at25° C./60% RH (Relative Humidity), with brown glass bottles as samplecontainer.

Test Release data 0 month 1 month 3 month Residual solvent: 7.5 7.6 7.67.1 % (w/w) ethanol % (w/w) Water 0.10 0.27 0.26 0.55

Example 7 Adsorption-Desorption Tests

About 23 mg of Form A were transferred into a VTI vapor sorptionanalyzer model SGA100 and the weight change with respect to theatmospheric humidity was recorded at the following conditions:

-   -   drying temperature: 40° C.    -   equilibrium: ≦0.05% in 5 min. or 60 min.    -   data interval: 0.05% or 2 min.    -   temperature: 25° C.    -   first cycle RH (%) adsorption: 5, 10, 20, 30, 40, 50, 60, 70,        80, 90, 95        -   RH (%) desorption: 95, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5    -   second cycle RH (%) adsorption: 5, 10, 20, 30, 40, 50, 60, 70,        80, 90, 95        -   RH (%) desorption: 95, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5

At the drying step about 0.6% weight loss was registered. The obtaineddried product was not hygroscopic, it adsorbed up to 0.7% water at highrelative humidity. During the desorption cycle a loss of weight of 1.4%was registered, this indicated that the product was losing ethanolate.The obtained product after ADS/DES was a mixture of ethanolate form andhydrated form.

The ADS/DES curve is collected in FIG. 18.

Adsorption-Desorption Hydration Tests

About 23 mg of Form A were transferred into a VTI vapor sorptionanalyzer model SGA100 and the weight change with respect to theatmospheric humidity was recorded at the following conditions:

-   -   equilibrium: ≦0.0005% in 5 min. or 90 min.    -   data interval: 0.05% or 2 min    -   temperature: 25° C.    -   cycle RH (%) adsorption/desorption: 5.95        -   repeat the cycle 11 times

At the end of this test a loss of weight of 5.2% was registered. Thiswas comparable with the TG result (TG 5.4% up to 80° C.). The ethanolateform was transferred into a hydrated form. The ADS/DES hydration testcurves are collected in FIG. 19.

Example 8

The stability of Form A was studied after storage of the compound in asample container with an inner cover made of single LD-PE (stringsealed), and outer cover made of PETP/Alu/PE (Moplast) heat sealed. Along term stability study at 25° C./60% RH, and an accelerated stabilitystudy at 40° C./75% RH, were performed for a period of 6 months, and thesamples analysed at different time points as shown in following tables.

TABLE 13 Long term stability at 25° C./60% RH Release tests RemarkSpecification data 0 month 1 month 3 month 6 month Polymorphism ° C.(onset) For information only 97.3 97.3 95.5 97.9 97.5 DSC ° C. max Forinformation only 104 104.2 103.5 104.2 104 Residual % (w/w) ethanol<=10.0%  6.71 6.31 6.33 6.40 6.33 solvents % (w/w) 2-propanol <=0.5%0.04 0.04 0.05 0.05 0.05 % (w/w) THF <=0.5% <0.01 <0.01 <0.01 <0.01<0.01 % (w/w) acetone <=0.5% <0.01 <0.01 <0.01 <0.01 <0.01 % (w/w)CH₂Cl₂ <=0.06%  <0.01 <0.01 <0.01 <0.01 <0.01 Water (KF) % (w/w) <=7.0%0.63 0.23 0.34 0.32 0.46 X-Ray For information only C C — — — powderdiffraction C: chrystal

TABLE 14 Accelerated stability at 40° C./75% RH Release 0 1 3 6 TestsRemark Specification data month month month month Polymorphism ° C.(onset) For information only 97.3 97.3 97.5 98.0 97.8 DSC ° C. max Forinformation only 104 104.2 103.4 1039 104.3 Residual % (w/w) ethanol<=10.0%  6.71 6.31 6.73 6.32 6.50 solvents % (w/w) 2-propanol <=0.5%0.04 0.04 0.05 0.05 0.05 % (w/w) THF <=0.5% <0.01 <0.01 <0.01 <0.01<0.01 % (w/w) acetone <=0.5% <0.01 <0.01 <0.01 <0.01 <0.01 % (w/w)CH₂Cl₂ <=0.06%  <0.01 <0.01 <0.01 <0.01 <0.01 Water (KF) % (w/w) <=7.0%0.63 0.23 0.37 0.34 0.42 X-Ray For information only C C — — — powderdiffraction

Form A exhibited chemical and crystallographic stability at theconditions mentioned in tables 13 and 14.

Example 9

The stability of Form A was studied after storage of the compound in asample container with an inner cover made of single LD-PE (stringsealed), and outer cover made of vapor loc bag (LPS) heat sealed. A longterm stability study at 25° C./60% RH, and an accelerated stabilitystudy at 40° C./75% RH, were performed for a period of 6 months, and thesamples analysed at different time points as shown in following tables.

TABLE 15 Long term stability at 25° C./60% RH Release Tests RemarkSpecification data 0 month 1 month 3 month 6 month Polymorphism ° C.(onset) For information only 97.3 97.3 96.3 96.2 98.5 DSC ° C. max Forinformation only 104 104.2 103.1 103.8 103.9 Residual % (w/w) ethanol<=10.0%  6.71 6.31 6.42 6.35 6.52 solvents % (w/w) 2-propanol <=0.5%0.04 0.04 0.06 0.05 0.05 % (w/w) THF <=0.5% <0.01 <0.01 <0.01 <0.01<0.01 % (w/w) acetone <=0.5% <0.01 <0.01 <0.01 <0.01 <0.01 % (w/w)CH₂Cl₂ <=0.06%  <0.01 <0.01 <0.01 <0.01 <0.01 Water (KF) % (w/w) <=7.0%0.63 0.23 0.32 0.38 0.49 X-Ray For information only C C — — — powderdiffraction

TABLE 16 Accelerated stability at 40° C./75% RH Release 0 1 3 6 TestsRemark Specification data month month month month Polymorphism ° C.(onset) For information only 97.3 97.3 97.8 97.5 97.9 DSC ° C. max Forinformation only 104 104.2 103.4 103.7 104.0 Residual % (w/w) ethanol<=10.0%  6.71 6.31 6.35 6.31 6.30 solvents % (w/w) 2-propanol <=0.5%0.04 0.04 0.06 0.05 0.05 % (w/w) THF <=0.5% <0.01 <0.01 <0.01 <0.01<0.01 % (w/w) acetone <=0.5% <0.01 <0.01 <0.01 <0.01 <0.01 % (w/w)CH₂Cl₂ <=0.06%  <0.01 <0.01 <0.01 <0.01 <0.01 Water (KF) % (w/w) <=7.0%0.63 0.23 0.31 0.36 0.51 X-Ray For information only C C — — — powderdiffraction

Form A exhibited chemical and crystallographic stability at theconditions mentioned in tables 15 and 16.

Example 10

For the purpose of chemical stability testing, Form A was stored for aperiod of 1, 4 and 8 weeks under different conditions. These conditionswere 40° C./75% RH, 50° C., RT/<5% RH, RT/56% RH, RT/75% RH and 0.3 daICH light. The compound was analysed after storage by HPLC and by visualinspection. The HPLC method used in this study was HPLC method 909. Theresults of the tests are reported in the following table.

TABLE 17 HPLC Sum of impurities Appearance Conditions 1 week 4 week 8week 1 week 4 weeks 8 weeks Reference 1.07 — — slightly-yellow — — 0.3da ICH light 1.01 — — slightly-yellow — — 40° C./75% RH 1.03 0.98 0.99slightly-yellow slightly-yellow slightly-yellow 50° C. 1.05 1.08 1.06slightly-yellow slightly-yellow slightly-yellow RT/<5% RH — 1.02 1.04 —slightly-yellow slightly-yellow RT/56% RH — 1.02 0.99 — slightly-yellowslightly-yellow RT/75% RH — 1.00 1.01 — slightly-yellow slightly-yellow

It was concluded that Form A is chemically stable after storage in allinvestigated conditions.

Example 11

Different fractions of Form B were characterized with thermogravimetry(TG), differential scanning calorimetry (DSC) and infrared spectroscopy(IR). The results of the tests are reported in the following table.

TABLE 18 TG % weight DSC change Max Extra Fractions <100° C. IR (° C.)(° C.) Form B fraction 1 5.65 Hydrate, Ref 69.1 — after ADS/DES 4.30±Hydrate, Ref, — — +amorphous Form B fraction 2 5.91 ~Hydrate, Ref 75.6— after 5 d 40° C./75% RH 3.56 ~Hydrate, Ref 74.1 — Form B fraction 33.13 ±Hydrate, Ref, 77.0 67.8 +amorphous after 5 d 40° C./75% RH 2.33±Hydrate, Ref, 77.4 62.8 +amorphous ~hydrate, Ref: identical withreference

Example 12

The adsorption and desorption of water at 25° C. at different conditionsof relative humidity was investigated on 38 mg of Form B. The weightchange as a function of relative humidity was registered. The resultsare displayed in FIG. 20. At the drying step about 5.6% weight loss wasregistered for Form B. The obtained dried product was hygroscopic, itadsorbed up to 6.8% water at high relative humidity. After thedesorption cycle about 1.2% water remained on the sample. The obtainedproduct after ADS/DES was a mixture of hydrate and amorphous product.

Example 13

Aqueous solubilities of Form B were measured in solvents with differentpH. An excess of the solute was equilibrated with the solvent at 20° C.for at least 24 hours. After removing the undissolved compound, theconcentration in solution was determined using UV spectrometry.

TABLE 19 Solvent Solubility (mg/100 ml solution) Water 10 (pH 5.1)Buffer pH 2 (citrate/HCl) 23 (pH 2.0) Buffer pH 3 (citrate/HCl) 13 (pH3.0) Buffer pH 4 (citrate/HCl) 12 (pH 4.0) 0.01N HCl 18 (pH 2.1) 0.1NHCl 150 (pH 1.1) 1.0N HCl 510 (pH 0.14)

Example 14

The stability of the crystal structure of Form B was studied afterstorage of the compound for a period of two weeks at room temperature(RT) under <5%, 56% and 75% relative humidity (RH), 50° C. and 40°C./75% RH. The samples were analyzed with thermogravimetry (TG),differential scanning calorimetry (DSC), infrared spectroscopy (IR) andX-ray diffraction (XRD). The results of the tests are reported in thefollowing table.

TABLE 20 TG DSC condition <100° C. <225° C. IR XRD Max (° C.) Appearance0 days 5.65 0.16 Ref Ref 69.1 slightly yellow-orange after ADS/DES 4.300.18 ≠Ref — — slightly yellow-orange RT/<5% RH 0.32 0.07 ≠Ref ≠Ref 71.2slightly yellow-orange RT/56% RH 5.71 0.25 ~Ref ~Ref 71.0 slightlyyellow-orange RT/75% RH 6.20 0.10 ~Ref ~Ref 71.5 slightly yellow-orange50° C. 0.23 0.06 ≠Ref ≠Ref 76.4 slightly yellow-orange 40° C. 75% RH5.77 0.07 ~Ref ±Ref 70.4 slightly yellow-orange ~Ref: identical withreference ±Ref: similar with reference ≠Ref: different with reference

Example 15

In the chemical stability test program Form B was stored for a period of1, 4 and 9 weeks under different conditions. These conditions were 40°C./75% RH, 50° C., RT/<5% RH, RT/56% RH, RT/75% RH and 0.3 da ICH light.The compound was analysed after storage by HPLC and by visualinspection. The HPLC method used in this study was HPLC method 909. Theresults of the tests are reported in the following table, from which itwas concluded that Form B is chemically stable.

TABLE 21 HPLC Sum of impurities Appearance Condition 1 week 4 week 9week 1 week 4 weeks 9 weeks Reference 1.35 — — lightly yellow- — —orange 0.3 da ICH 1.30 — — light-orange — — light 40° C./75% RH 1.431.38 1.41 lightly yellow- Orange light-orange orange 50° C. 1.46 1.501.46 lightly yellow- light-orange light-orange orange RT/<50% RH — 1.481.37 — light-orange light-orange RT/56% RH — 1.11 1.35 — lightly yellow-light-orange orange RT/75% RH — 1.34 1.29 — light-orange light-orange

Example 16

Form K was prepared by adding neat methanesulfonic acid to a solution ofForm A in THF at r.t. Form K was subsequently mixed with alkali halideand pressed to a pellet (Ph. Eur.) and analyzed by Infrared spectrometry(IR) at the following conditions:

-   -   apparatus: Nicolet Magna 560 FTIR spectrophotometer    -   number of scans: 32    -   resolution: 1 cm⁻¹    -   wavelength range: 4000 to 400 cm⁻¹    -   baseline correction: yes    -   detector: DTGS with KBr windows    -   beamsplitter: Ge on KBr    -   alkali halide: KBr (potassium bromide)

The IR spectrum of Form K, as shown in FIG. 21, reflects the vibrationalmodes of the molecular structure of the mesylate solvate as acrystalline product.

TABLE 22 Wavenumbers (cm⁻¹) and relative intensities of absorption bands(¹) 3362m, 3064w 2985m, 2964m, 2906m, 2873m, 2632w, 2585w 1687s, 1627w,1601w 1554m, 1495m, 1480w, 1470w, 1452w, 1443w, 1421w 1383w, 1373w,1369w, 1345m, 1324m, 1314m, 1299w, 1268m, 1245m, 1221m, 1202s 1190s,1166vs, 1122m, 1091m, 1077m, 1051s, 1043s, 1023m, 1002m 992m, 969w,943w, 912w, 888w, 867vw, 836w, 813vw 773m, 754w, 743m, 711w, 700m, 658m,634w 581w, 556m, 505w, 472vw, 452vw, 435vw, 417vw (¹) vs = very strong,s = strong, m = medium, w = weak, vw = very weak, br = broad

Example 17

Form K was transferred to a glass capillary cell and analyzed by Ramanspectrometry at the following conditions:

-   -   Raman mode: Nondispersive Raman    -   apparatus: Nicolet FT-Raman module    -   number of scans: 64    -   resolution: 4 cm⁻¹    -   wavelength range: 3700 to 100 cm⁻¹    -   laser: Nd:YVO4    -   laser frequency: 1064 cm⁻¹    -   detector: InGaAs    -   beamsplitter: CaF₂    -   sample geometry: 180° reflective    -   polarization: no

The Raman spectrum of Form K, as shown in FIG. 22, reflects thevibrational modes of the molecular structure of the mesylate as acrystalline product.

TABLE 23 Wavenumbers (cm⁻¹) and relative intensities of absorption bands(¹) 3080m, 3068m, 3059m, 3043w, 3022w, 3006m 2989s, 2978s, 2962s,2933vs, 2906m, 2871m 1685vw, 1628w, 1603s, 1585w, 1495w, 1479w, 1466w,1450m, 1423w 1381w, 1346w, 1336w, 1313w, 1290w, 1271w, 1244w, 1230w,1209m 1190w, 1182m, 1163vs, 1122w, 1105w, 1090m, 1049vs, 1032w, 1003s968w, 955w, 941w, 914w, 897w, 877w, 866w, 845w, 823m, 814m 783m, 771m,742w, 658w, 634m, 621w 577w, 561m, 534w, 524w, 497w, 451w, 436w 337w,308w, 287m, 247w, 206w, 162m, 129m (¹) vs = very strong, s = strong, m =medium, w = weak, vw = very weak

Example 18

About 3 mg of Form K were transferred into a standard aluminiumTA-Instrument sample pan. The sample pan was closed with the appropriatecover and the DSC curve recorded on a TA-Instruments Q1000 MTDSCequipped with a RCS cooling unit, at the following conditions:

-   -   initial temperature: 25° C.    -   heating rate: 10° C./min    -   final temperature: 200° C.    -   nitrogen flow: 50 ml/min

The DSC curve as depicted in FIG. 23, shows the melting withdecomposition of a crystalline product. The melting of Form K occurs at158.4° C. Due to the decomposition, the heat of fusion calculation canonly be used to indicate the crystalline property of the product.

Example 19

Form K was transferred into an aluminum sample pan. The TG curve wasrecorded on a TA Instruments Hi-Res TGA 2950 thermogravimeter at thefollowing conditions:

-   -   initial temperature: room temperature    -   heating rate: 20° C./min    -   resolution factor: 4    -   final condition: 300° C. or <80[(w/w) %]

The TG curve is exhibited in FIG. 24. The loss of weight of around O₂%up to 60° C. was due to the evaporation of solvent. The loss of weightat temperatures above 140° C. was due to the evaporation anddecomposition of the product.

Example 20 Adsorption-Desorption

About 21 mg of Form K were transferred into a VT1 vapor sorptionanalyzer model SGA100 and the weight change with respect to theatmospheric humidity was recorded at the following conditions:

-   drying temperature: 40° C.-   equilibrium: 5.0.05% in 5 min. or 60 min.-   data interval: 0.05% or 2.0 min.-   temperature: 25° C.-   first cycle RH (%) adsorption: 5, 10, 20, 30, 40, 50, 60, 70, 80,    90, 95    -   RH (%) desorption: 95, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5-   second cycle RH (%) adsorption: 5, 10, 20, 30, 40, 50, 60, 70, 80,    90, 95    -   RH (%) desorption: 95, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5

The Adsorption-Desorption isotherm is shown in FIG. 25. Form K ishygroscopic. At the initial drying step a loss of weight of 0.3% wasregistered, comparable to the TG result. Form K adsorbed up to 1.5%water at high relative humidity. The product dried completely during thedesorption cycle.

A different study of the adsorption and desorption of water by Form K at25° C. at different conditions of relative humidity was investigated onan amount of about 18 mg of the mesylate solvate. The weight change as afunction of relative humidity was registered. The result is displayed inFIG. 26.

At the drying step about 0.6% weight loss is registered for Form K. Theobtained dried product is slightly hygroscopic, it adsorbed up to 1.7%water at high relative humidity. The product dried completely during thedesorption cycle.

Example 21

Aqueous solubilities of Form K were measured in solvents with differentpH. An excess of the solute was equilibrated with the solvent at 20° C.for at least 48 hours. After removing the undissolved compound, theconcentration in solution was determined using UV spectrometry.

TABLE 24 Solvent Solubility (mg/100 ml solution) Water 19 (pH 3.3)Buffer pH 2 (citrate/HCl) 21 (pH 2.0) Buffer pH 3 (citrate/HCl) 12 (pH3.0) Buffer pH 4 (citrate/HCl) 11 (pH 4.0) 0.01N HCl 24 (pH 2.0) 20%HPβCD in water 2100 (pH 1.6)

Example 22

The stability of the crystal structure of Form K batch 1 was studiedafter storage of the compound for a period of four weeks at roomtemperature (RT) under 75% relative humidity (RH), 50° C. and 40° C./75%RH. The stability of the crystal structure of Form K batch 2 was studiedafter storage of the compound for a period of four weeks at roomtemperature (RT) under <5%, 56% and 75% relative humidity (RH), 50° C.and 40° C./75% RH. The samples were analyzed with thermogravimetry (TG),differential scanning calorimetry (DSC) and infrared spectroscopy (IR).The results of the tests are reported in the following table.

TABLE 25 DSC TG Max Extra compound conditions <80° C. <125° C. IR (° C.)(° C.) Appearance Form K 0 days 0.47 0.15 Ref 143.7 — slightly orangeBatch 1 RT/75% RH 2.87 0.19 ≠Ref 146.6 64.3 slightly orange 50° C. 0.320.14 ~Ref 140.6 45.6 orange 40° C./75% RH 1.48 3.71 — — — brown oil FormK 0 days 0.16 0.11 Ref 155.8 — slightly orange Batch 2 RT/<5% RH 0.000.03 ~Ref 156.9 — slightly orange RT/56% RH 0.27 0.03 ±Ref 154.6 —slightly orange RT/75% RH 1.82 0.07 ≠Ref 149.2 67.0 slightly orange 50°C. 0.12 0.12 ~Ref 156.8 — slightly orange 40° C./75% RH 3.26 3.08 — — —brown oil ~Ref: identical with reference ±Ref: similar with reference≠Ref: different with reference

Example 23

In the chemical stability test program Form K batch 1 was stored for aperiod of 1 and 4 weeks under different conditions. These conditionswere 40° C./75% RH, 50° C., RT/75% RH and 0.3 da ICH light. Form K batch2 was also stored for a period of 1 and 4 weeks under differentconditions. These conditions were 40° C./75% RH, 50° C., RT/<5% RH,RT/56% RH, RT/75% RH and 0.3 da ICH light. The compound was analysedafter storage by HPLC and by visual inspection. The HPLC method used inthis study was HPLC method 909. The results of the tests are reported inthe following table.

TABLE 26 HPLC Sum of impurities appearance compound conditions 1 week 4weeks 1 week 4 weeks Form K batch 1 Reference 3.57 — slightly-orange —0.3 da ICH light 2.93 — slightly-orange — 40° C./75% RH 5.36 >90*    slightly-orange brown oil 50° C. 3.99 27.53  slightly-orange orangeRT/75% RH — 3.61 — slightly-orange Form K Batch 2 Reference 1.50 —slightly-orange — 0.3 da ICH light 1.17 — slightly-orange — 40° C./75%RH 1.75 >85*     slightly-orange brown oil 50° C. 1.46 1.25slightly-orange slightly-orange RT/<5% RH — 1.58 — slightly-orangeRT/56% RH — 1.45 — slightly-orange RT/75% RH — 1.46 — slightly-orange

Example 24

A randomized, placebo-controlled, double-blind, multiple dose escalationtrial was performed to examine the safety, tolerability andpharmacokinetics of Form A after oral administration twice or threetimes daily, in healthy subjects. Four dosages of Form A (400 mg b.i.d.,800 mg b.i.d., 800 mg t.i.d., and 1200 mg t.i.d.) were tested in 4panels of 9 healthy subjects. Within each panel, 6 subjects were treatedwith Form A and 3 subjects with placebo for 13 days with a single intakein the morning of day 14. (b.i.d.=twice daily, t.i.d.=three timesdaily).

Form A was readily absorbed and concentration-time profiles of Form Aafter repeated dosing were dependent on the dose administered.Steady-state plasma concentrations were reached generally within 3 days,although C_(0h) (conc. at administration time) and AUC_(24h) (area underde curve or bioavailability) slightly decreased over time at all doselevels. AUC_(24h) and C_(ss,av) (conc. at average steady-state) weredose-proportional (daily dose) at 400 mg b.i.d., 800 mg t.i.d. and 1200mg t.i.d., but was more than dose-proportional at 800 mg b.i.d. C_(max)(maximum conc.) was dose-proportional with respect to dose per intake.Less than 2% of unchanged Form A was excreted in the urine at all doselevels.

1-14. (canceled)
 15. An ethanolate solvate of the compound(3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl(1S,2R)-3-[[(4-aminophenyl)sulfonyl](isobutyl)amino]-1-benzyl-2-hydroxypropylcarbamate,in which the ratio of compound to ethanol is about 1:1.